1. Field of Invention
This application relates to a method for making site-specific protein modifications, and more particularly to modifications of protein molecules having at least two non-terminal cysteine residues; and to products comprising protein molecules so modified.
2. Discussion of Related Art
The contents of all references, including articles, published patent applications and patents referred to anywhere in this specification are hereby incorporated by reference.
Biochemical processes often exhibit a large degree of heterogeneity. A prominent example is the myriad of successive conformations experienced by an unfolded (denatured) protein along its folding trajectory towards its native state.1-3 Classical ensemble methods yield only mean values, averaged over large ensembles of molecules. Single molecule experiments, on the other hand, allow the examination of each molecule individually. Meaningful information about the microscopic distribution of conformations, trajectories and sequences of events can be obtained that are hidden on ensemble level.4-9 
Fluorescence resonance energy transfer (FRET) between a single donor (D) fluorophore and a complementary single acceptor (A) fluorophore (single-pair FRET, or spFRET) is a particularly powerful and sensitive method for monitoring protein folding reactions at single molecule resolution.10-14 The FRET-efficiency E is a sensitive function of the D/A- distance R, as E=[1+(R/R0)6]-1. R0 is a constant that corresponds to a D/A-distance at which E=50%.15 Because of its dependence on the distance R, spFRET can be used as a distance ruler to track intrachain-conformational dynamics in polypeptide chains in the 2 to 8 nm range.16 
A critical component in a single molecule spFRET protein folding experiment is the ability to label a polypeptide chain with a unique D/A-pair in a controlled and site-specific way. In the past, single molecule spFRET folding studies have been performed with chemically synthesized polypeptides.4,5,17 Chemical synthesis of polypeptides has the advantage that side chain protecting groups can be exploited to facilitate site-specific two-color labeling, but its extension to 3-color labeling18-20 or the labeling of proteins of more than 100 amino acids in length are difficult to achieve.
Recombinant expression of proteins offers more flexibility with respect to chain size. Cysteine (Cys) residues are statistically underrepresented in protein sequences, and many proteins are either devoid of Cys or intrinsic Cys can be removed by site-directed mutagenesis. A unique pair of Cys can then be (re)introduced into the protein at carefully selected surface accessible positions for conjugation with thiol-specific maleimide-functionalized fluorophores. For stoichiometric labeling of double-Cys proteins with a D/A-pair, a two step protocol is usually employed.6,7,21,22 First, the protein is reacted with a single fluorophore, added at stoichiometric ratio to minimize double labeling. Singly modified protein molecules are then separated chromatographically from unreacted or doubly-labeled molecules and reacted with the second, complementary, dye. Unfortunately, this two-step sequential labeling is not strictly site-specific. Unless the dye-accessibility of the two thiol-groups differ drastically, the first added fluorophore can be attached to either of the two sulfhydryl groups, giving rise to mixtures of D/A-labeled molecules and the dye-permutated, A/D-analogues.22 Such mixtures can lead to unwanted sample heterogeneity, as the conjugated dyes can exert a positional-dependent perturbation of the folding free energy of the modified protein. Also, heterogeneities in photophysical properties of the fluorophores due to different local environments (local charge, pH, or hydrophobicity) could complicate the interpretation of spFRET measurements.23,23 Lastly, strict site-specificity of labeling is absolutely mandatory for more sophisticated three- or multi-color FRET experiments.18-20 
To increase the site-specificity of sequential labeling, labeling chemistries have been developed that selectively modify N-terminal Cys residues. For example, N-terminal Cys specifically react with thioester-moieties into a stable amide bond.25-27 This chemistry has been exploited by Schuler and Pannell28 to label a short synthetic model peptide at the N-terminus using a commercial fluorophore chemically modified with a thiobenzylester functionality. Other strategies involve the oxidation of an N-terminal serine (Ser) or threonine (Thr) to the corresponding aldehyde and subsequent coupling with fluorophore containing hydrazine, alkoxyamine or hydrazide functionalities,29 or the specific reaction of an N-terminal Cys with aldehydes into thiazolidines, a reaction that has been utilized to label and immobilize peptides and proteins.30-33 
Recently, Schultz and colleagues reported a novel strategy for site-specific incorporation of non-natural amino acids into proteins in vivo in response to the amber stop codon using genetically modified orthogonal t-RNA/t-RNA synthetase pairs with altered amino acid specificities. Incorporation of non-natural amino acids with keto or azide functionalities into soluble cytoplasmic as well as membrane proteins have been achieved, with excellent yields and high fidelity.34-37 The unique chemistry of the keto and azide groups can be used for site-specific dye-conjugation either directly in vivo (e.g. by addition of a hydrazine-fiunctionalized dye to the growth medium to label the non-natural keto-group) or in vitro using purified protein samples. Although very powerful, this technique is not yet broadly available to the scientific community. There thus remains a need for at least improved techniques for site-specific labeling of proteins.